Oxygenated Scaffolds for Pancreatic Endocrine Differentiation from Induced Pluripotent Stem Cells

Abstract

A 3D microenvironment is known to endorse pancreatic islet development from human induced pluripotent stem cells (iPSCs). However, oxygen supply becomes a limiting factor in a scaffold culture. In this study, oxygen-releasing biomaterials are fabricated and an oxygenated scaffold culture platform is developed to offer a better oxygen supply during 3D iPSC pancreatic differentiation. It is found that the oxygenation does not alter the scaffold's mechanical properties. The in situ oxygenation improves oxygen tension within the scaffolds. The unique 3D differentiation system enables the generation of islet organoids with enhanced expression of islet signature genes and proteins. Additionally, it is discovered that the oxygenation at the early stage of differentiation has more profound impacts on islet development from iPSCs. More C-peptide⁺ /MAFA⁺ β and glucagon⁺ /MAFB⁺ α cells formed in the iPSC-derived islet organoids generated under oxygenated conditions, suggesting enhanced maturation of the organoids. Furthermore, the oxygenated 3D cultures improve islet organoids' sensitivity to glucose for insulin secretion. It is herein demonstrated that the oxygenated scaffold culture empowers iPSC islet differentiation to generate clinically relevant tissues for diabetes research and treatment.

Keywords: in-situ oxygenation; induced pluripotent stem cells; insulin; islet organoids; pancreatic differentiation.

Figure 1.. Oxygenated scaffolds developed for iPSC pancreatic differentiation.

A) Oxygen release kinetics of the oxygenators. Results were from three independent experiments and shown as mean ± SD. B) Schematic diagrams of cell-laden oxygenated scaffolds. C,D) Cell viability in the non-oxygenated (C) and oxygenated (D) scaffolds at 24 h post seeding. E,F) Cell viability in the non-oxygenated (E) and oxygenated (F) scaffolds at 48 h post seeding. G) Cell viability in the non-oxygenated (NOXY) and oxygenated (OXY) scaffolds at 24 and 48 h post cell seeding, quantified by trypan blue assay. Cell viability under oxygenated condition was normalized to that under non-oxygenated condition. Results were from three independent experiments and shown as mean ± SD.

Figure 2.. SEM images of acellular collagen scaffolds in the presence or absence of the oxygenators.

A) Yellow area indicates the space left behind after the oxygenator was removed. The image was taken at 100×. B) Grape-like structures in the presence of the oxygenator at 5000×. C) Grape-like structures in the absence of the oxygenator at 5000×. D–G) Scaffolds in the absence of the oxygenators. H–K) Scaffolds in the presence of the oxygenators but the areas have no direct interaction with the oxygenator material. L–O) Scaffolds in the presence of the oxygenators in the areas next to the oxygenator material. Magnifications: (D, H, and L): 5K; (E, I, and M): 10K; (F, J, and N): 20K; (G, K, and O): 40K. P) Estimation of mean value of fibrous diameter as measured by ImageJ. n = 259 for no oxygenator group; n = 138 for the areas without direct contact to the oxygenator; n = 114 for the areas with direct contact to the oxygenator. *: p < 0.001. Q) Estimation of mean value of pore size as measured by ImageJ. n = 328 for no oxygenator group; n = 281 for the group without direct contact to the oxygenator; n = 182 for the group with direct contact to the oxygenator. *: p < 0.005, **: p < 0.001. ns: not significant.

Figure 3.. Microstructure of iPSC-oxygenator embedded scaffolds.

A,B) Cell-laden scaffolds in the absence of oxygenators. C,D) Cells next to the oxygenators. E,F) Cells away from the oxygenators. G) Diameters of the collagen fibers measured by ImageJ. n = 72 for the group without oxygenator; n = 103 for the areas without direct contact to the oxygenator (OXY). n = 91 for the areas with direct contact to the oxygenator. ***: p < 0.001. H) Pore sizes of collagen f networks ascertained by ImageJ. n = 248 for no oxygenator group; n = 331 for the areas without direct contact to the oxygenator; n = 354 for the areas with direct contact to the oxygenator. *: p < 0.05, **: p < 0.01. ns: not significant. Scale bars: 2 μm.

Figure 4.. Mechanical properties of non-oxygenated and oxygenated acellular scaffolds.

A) G′ and G″ moduli at various angular frequencies of the scaffolds with and without in site oxygenation. B) Stiffness of oxygenated and non-oxygenated scaffolds determined at 1% strain and 12.6 rad s⁻¹. C) Viscosity of oxygenated and non-oxygenated scaffolds determined at various angular frequencies. D) Viscosity of oxygenated and non-oxygenated scaffolds measured at 1% strain and 0.1 rad s⁻¹. Results were from three independent experiments and shown as mean ± SD. ns: not significant.

Figure 5.. Oxygen tension in oxygenated (OXY) and non-oxygenated (NOXY) scaffolds at the end of iPSC definitive endoderm differentiation.

A) A schematic diagram of an iPSC differentiation protocol with key molecules applied for definitive endoderm development in the oxygenated scaffolds. NaB: sodium butyrate. BSA: bovine serum albumin. B) Oxygen tension at the surface (n = 12) and the bottom (n = 12) of the scaffolds measured by a well-established needle-type microsensor for top of the scaffolds and sensor foil for the bottom of the scaffolds. Results were obtained from four independent experiments and shown as mean ± SD. ***p < 0.01; ns: not significant. C) The oxygen sensor foil was placed under the cell-laden scaffold. The portable detector was mounted underneath the plate to measure the distribution of O₂ tension at the bottom of a scaffold. D) Image of a color-coded oxygen map generated at the bottom of the scaffold in the absence of the oxygenator (NOXY). E) Oxygen tension across the center line of the scaffold indicated by the line in (C). F) Distribution of oxygen tension at the bottom in the presence of the oxygenator (OXY). G) Oxygen tension across the center line of the scaffold indicated by the line in (E). (H,I) Spatial distribution of iPSC-derived definitive endoderm inside the scaffolds viewing from top to bottom. Cells were stained with DAPI (blue).

Figure 6.. Oxygen tension within the non-oxygenated and oxygenated scaffolds at the end of iPSC pancreatic progenitor differentiation.

A) A schematic diagram of an iPSC differentiation protocol with key molecules applied for pancreatic progenitor development in oxygenated scaffolds. NOXY: without oxygenation. OXY: with oxygenation. RA: retinoic acid. KGF: keratinocyte growth factor. LDN: LDN193189. ILV: (−)-indolactam V. VC: ascorbic acid. T3: 3,3′,5-triiodo-l-thyronine sodium salt. Rep: Repsox. B) Percentage of oxygen tension at the surface (n = 18) and the bottom (n = 16) of the scaffolds. Results were shown as mean ± SD. ***p < 0.001. C) Distribution of oxygen tension at the bottom of a non-oxygenated scaffold. D) Oxygen tension across the center line of the scaffold indicated by the line in (C). E) Distribution of oxygen tension at the bottom of an oxygenated scaffold. F) Oxygen tension across the center line of the scaffold indicated by the line in (E). G,H) Spatial distribution of iPSC-derived pancreatic progenitors inside the scaffolds viewing from top to bottom. Cells were stained with DAPI (blue).

Figure 7.. Oxygenated 3D microenvironment promotes the formation of pancreatic progenitors and subsequent pancreatic endocrine development from iPSCs.

A) A 29-day differentiation protocol with key molecules applied in stepwise differentiation procedures for islet organoid development within scaffolds. NOXY: without oxygenation. OXY: with in-situ oxygenation. Nic: nicotinamide. N-Cys: N-acetyl cysteine. Tro: trolox. γ-sec: γ-secretase inhibitor XX. B) Marker gene expressions of pancreatic progenitors under various microenvironments. Results were from five independent experiments and shown as mean ± SD. Gene expression was normalized to IMR90 cells. Human pancreas (hPancreas) served as a positive control. 3D-3-NOXY: 3 mm thickness of the scaffold without oxygenation. 3D-3-OXY: 3 mm thickness of the scaffold with oxygenation. 3D-5-NOXY: 5 mm thickness of the scaffold without oxygenation. 3D-5-OXY: 5 mm thickness of the scaffold with oxygenation. Different letters denote significant differences from one another. C) Gene expression of key markers of islet cells. Results were from four independent experiments and shown as mean ± SD. Groups with different letters denote significant differences from one another. *p < 0.05, **p < 0.01, ***p < 0.001. Significant differences in 3D groups compared to 2D cultures were determined and represented as #: p < 0.05; ## p < 0.01; and ### p < 0.001.

Figure 8.. Oxygenation promotes organogenesis and morphogenesis of iPSC-derived islet organoids.

A) A comparison of gene expression of NKX6.1, INS, and GCG with and without the replacement of oxygenators at day 19 during the differentiation. Results were from three independent experiments and shown as mean ± SD. B) A comparison of insulin stimulation index of iPSC-derived islet organoids generated under indicated conditions. Results were from four independent experiments and shown as mean ± SD. 3D-3-NOXY: 3 mm thickness of the scaffold without oxygenation. 3D-3-OXY: 3 mm thickness of the scaffold with oxygenation. Human islets (hIslet) were used as a positive control. *: p < 0.05, and NS: nonsignificant. C–I) Enhancement of organogenesis and morphogenesis of islet organoids generated in oxygenated scaffolds. At the end of differentiation, the organoids were immunofluorescently labeled for (C) C-peptide (CP, red) and glucagon (GCG, green); (E) somatostatin (SST, green) and pancreatic polypeptide (PPY, red); (G) MAFA (green) and CP (red); and (I) MAFB (green) and GCG (red). Cells were counterstained with DAPI (blue). Scale bars, 50 μm. D,F,H,J) The organoid structures generated were semi-quantitatively analyzed using ImageJ to estimate approximate populations of cell subtypes. D) n = 25 images from five independent differentiation experiments for oxygenated scaffolds; n = 12 images from three independent differentiation experiments for non-oxygenated scaffolds. F,H,J) n = 6–10 images per individual condition from three independent experiments. Results were shown as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. ns: not significant.